FIGURE 5.

Gle1 regulates Ded1 in AUG start site selection. A, wild-type (WT), gle1–4, ded1–120, and gle1–4 ded1–120 strains were transformed with L0- and L2-luciferase reporters (reporter mRNAs diagrammed at left). Strains were grown overnight at 27.5 °C in selective media, then shifted to 23 °C for 4 h or not. Luciferase assays were performed on cell lysates, and scanning efficiency was calculated by the ratio of activity from L2-LUC to L0-LUC normalized for cell number. B, indicated strains were transformed with uORF1- and uORF1x-GCN4-lacZ reporters (diagram at left), and cultures were diluted in selective media. Following growth overnight at 23 °C or 30 °C, β-galactosidase assays were performed. Leaky scanning activity was determined as a percent ratio of uORF1x to uORF1 β-galactosidase activity. *, p < 0.05 versus WT; **, p < 0.01 versus WT; +, p < 0.05 versus ded1–120 strain. C, indicated strains were transformed with HIS4-lacZ-AUG and -UUG reporters (diagram at left), and cultures were diluted in selective media. Following growth overnight at 23 °C or 30 °C, β-galactosidase assays were performed. Start site fidelity was determined as a percent ratio of HIS4-UUG to HIS4-AUG β-galactosidase activity. *, p < 0.05 versus WT; **, p < 0.01 versus WT; +, p < 0.05 versus gle1–4 strain. For all panels, n = 3 to 10 for mean and S. E.